A technique for highly selective and sensitive analysis of drugs in serum and urine will be developed. The technique, Phase-Resolved Fluoroimmunoassay (PRFIA), is a homogeneous immunoassay technique which requires no separation of free from antibody-bound labelled antigen. Instead, the free labelled antigen is distinguished from the antibody-bound labelled antigen on the basis of the difference in fluorescence lifetimes of the fluorescent label in the two different microenvironments. Phase-resolved fluorescence spectroscopy (PRFS), in which phase-sensitive detection is used to selectively measure the fluorescence of species excited with sinusoidally-modulated light, will be used to simultaneously determine the percents of free and bound labelled antigen in sample and standard solutions. Interference due to bilirubin fluorescence in serum can be eliminated by treating the bilirubin as a separate component in the solution and resolving its contribution along with those of the free and bound labelled antigen. This eliminates the need for subtraction of a serum blank, even for icteric sera. Application of PRFIA to analysis of urine and of whole blood will also be investigated, treating other matrix interferents in a similar manner. The PRFIA technique will be optimized for determination of phenobarbital. Methods for other abused drugs, including tetrahydrocannabinol, phencyclidine (PCP), heroin, cocaine, opiates and others, will then be fully developed. Initial studies for the proposed project are currently funded by NIH ADAMHA Small Grants Program. Preliminary results of these studies will be described, including the use of PRFS to eliminate bilirubin interference in measurement of fluorescein, and initial results for PRFIA of phenobarbital. Advantages of the proposed PRFIA in comparison with other homogeneous immunoassay techniques include procedural simplicity, minimal reagent requirements, and a multicomponent approach which enables elimination of interference from sample matrix fluorophores.